WO2020098517A1 - Use of 4-(benzselenazol-2-yl)arylamine compound in treating stomach cancer or intestinal cancer - Google Patents

Abstract

Disclosed is a use of a 4-(benzselenazol-2-yl)arylamine compound in treating stomach cancer or intestinal cancer, wherein the compound is a compound as shown in general formula I or a pharmaceutically acceptable salt thereof. The present invention finds a new use of a 4-(benzselenazol-2-yl)arylamine compound in the treatment of diseases, and provides a potential medicine for the treatment of stomach cancer or intestinal cancer.

Description

Use of 4- (benzoselenazol-2-yl) arylamine compounds for treating gastric cancer or intestinal cancer Technical field

The invention belongs to the field of medicinal chemistry, and specifically relates to the use of a class of 4- (benzoselazol-2-yl) aromatic amine compounds in the treatment of gastric cancer or intestinal cancer.

Background technique

Gastric cancer is one of the most common malignant tumors of the digestive system. Worldwide, the incidence of gastric cancer ranks 5th among malignant tumors, and the mortality rate ranks 3rd among malignant tumors. According to data released by the International Cancer Research Center (IARC), there were 951,600 new cases of gastric cancer in the world in 2012 (including about twice as many male cases as women) and 723,100 deaths. According to statistics, the incidence of gastric cancer is highest in East Asia (especially South Korea, Mongolia, Japan, and China), followed by Central, Eastern, and South America in Europe, and the lowest in North America and most of Africa (Torre LA, Bray F, Siegel RL, et al.Global cancers, statisticals, 2012. Ca Cancer, Journal for Clinicians, 2015, 65 (2): 87-108; IARC. All Cancers (excluding non-melanoma skins cancer) Estimated Incidence, Mortality and Prevalence Worldwide Wider 2012; Herrero R , Park JY, Forman D. The Fight Against Against Gastric – Cancer – the IARC Working – Group report – Best Practice and Research – Clinical Gastroenterology, 2014, 28 (6): 1107-1114; Carcas LP. Gastric Cancer, Review. Journal of Carcinogenesis, 2014, 19 13-14).

According to statistics, the incidence of gastric cancer in China ranks second among malignant tumors, and the number of new cases and deaths each year exceeds 40% of the world (Torre LA, Siegel RL, Ward EM, et al. Global Cancer Incidence and Mortality Rates and Trends-an Update. Cancer Epidemiology and Prevention Biomarkers, 2016, 25 (1): 16-27); and about 352,300 patients die of gastric cancer each year (Layke JC, Lopez PP. Gastric cancer: diagnosis and treatment options. Chinese General Practice, 2015, 18 (3): 248-249).

The pathogenesis of gastric cancer has not yet been fully elucidated. It is mainly believed that the incidence of gastric cancer is related to factors such as dietary habits, Helicobacter pylori (Hp) infection, serum pepsinogen (PG), genetic factors, metabolic syndrome and psychological stress. . Helicobacter pylori may be the biggest predisposing factor for gastric cancer. About 90% of new non-cardia gastric cancer cases are related to this (Chang M, Zhang JC, Zhou Q, et al. Research Progress of of Clinical Epidemiology of Gastric Cancer. of Gastroenterology & Hepatology, 2017, 26 (9): 966-969).

At present, the treatment of gastric cancer is limited, and surgical resection is still the only clinical method to cure gastric cancer. For early gastric cancer, although it is not necessary to receive chemotherapy in principle after surgery, patients still need chemotherapy if they: (1) are younger than 40 years old; (2) there are multiple lesions; (3) the lesion area is greater than 5cm ² ; (4) The malignant degree of pathological types is high (He Aqing, Zhang Chengwu. Discussion on the diagnosis and treatment of gastric cancer. The World's Latest Medical Information Digest, 2018, 18 (14): 55).

Due to the incomplete gastric cancer screening system in China, the early diagnosis rate of gastric cancer is low, so most patients are already in advanced gastric cancer or advanced gastric cancer (Advanced Gastric Cancer) at the time of diagnosis. At this time, simple surgical treatment often fails to achieve good results, and the postoperative recurrence and metastasis rate is high; some patients have large tumors that cannot be operated on, and neoadjuvant chemotherapy must be given until the tumor volume is reduced before surgery can be performed. Studies have shown that for advanced gastric cancer, combined surgery can not only bring about survival advantages, and the overall survival of some patients is even lower than that of simple chemotherapy (Gastrectomy plus Chemotherapy vs Chemotherapy Alone for Advanced Gastric Cancer with Single Non-curable Factor (REGATTA) : aPhase3, RandomizedControlledTrial.LancetOncology, 2016,17 (3): 309-318). Therefore, chemotherapy-based comprehensive therapy is the main treatment for patients with advanced gastric cancer, which can improve the prognosis of patients and improve the quality of life of patients.

At present, the clinical cytotoxic drugs used for advanced gastric cancer chemotherapy mainly include four categories: (1) oral fluorouracil, such as capecitabine, tegio; (2) taxanes, such as paclitaxel, docetaxel ; (3) Third-generation platinums, such as oxaliplatin; (4) Topoisomerase I inhibitors, such as irinotecan. In addition, the marketed molecular targeted drugs mainly include human epidermal growth factor receptor 2 (HER2) antibodies, such as trastuzumab, vascular endothelial growth factor receptor (VEGFR-2) antibodies, such as ramucirumab, And the small molecule tyrosinase inhibitor Apatinib, etc. targeting VEGFR-2 (Li Kaichun, Li Ping. The choice of drugs for the treatment of advanced gastric cancer. Pharmaceutical Services and Research, 2018, 18 (1): 1-5 ).

Although the combination of chemotherapy for gastric cancer is the standard treatment recommended by the NCCN guidelines (NCCN Clinical Practice Guideline Oncology: Gastric Cancer (2015 Version)), the effect has reached a bottleneck, and the patient's survival benefit cannot be increased more, while , Patients will suffer from cytotoxicity of chemotherapy drugs such as leukocytes and thrombocytopenia and other toxic side effects, resulting in further deterioration of the patient's physical condition. In addition, gastric cancer is a highly heterogeneous disease driven by multiple genetic mutations and epigenetic abnormalities, so the development of targeted drugs has lagged, and existing drugs have poor targeting and low sensitivity, such as trastuzumab Only effective for about 20% of HER2-positive patients in gastric cancer patients (Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in Combination with Chemotherapy versus Chemotherapy Alone for Treatment of HER2-positive Advanced Gas- ToGA): aPhase 3, Open-label, Randomized Controlled Trial. Lancet, 2010, 376 (6): 687-697). Therefore, neither current chemotherapy nor targeted therapy can effectively combat gastric cancer, and increase the survival benefit of patients.

The incidence and mortality of gastric cancer in China is at a relatively high level in the world, which has seriously endangered the health of residents and caused a heavy burden on families and society. Due to the high heterogeneity of gastric cancer and the current lack of clinical treatment, there is an urgent need to develop new Treatment methods or drugs to cope with the increasing status of gastric cancer morbidity and mortality.

Colorectal cancer is the most common malignant tumor of the digestive system, and its global incidence ranks third in men and second in women (Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA, Cancer, Journal of Clinicians, 2015, 65 (2): 87-108). According to 2012 statistics, there are about 1.4 million new cases of colorectal cancer and 693,900 cases of colorectal cancer deaths worldwide. The regions with the highest incidence are in Australia and New Zealand, and Europe and North America are also high-incidence areas (Torre LA, Siegel RL, Ward EM, et.al. Global Cancer Incidence and Mortality Rates and Trends-an Update. Cancer Epidemiology and Prevention Biomarkers, 2016, 25 (1): 16-27). Colon cancer is the third most common cancer in the United States, with as many as 130,000 new cases of colon cancer diagnosed each year (Birt DF, Phillips GJ. Diet, Genes, and Microbes: Complexities of Colon Cancer Prevention. Toxicologic Pathology, 2014, 42: 182- 188). Because the early diagnosis rate of colon cancer is very low, the vast majority of patients are diagnosed in the middle and late stages, which makes it difficult for radical surgery. Its 5-year survival rate is only 50% -60% (Wang Zhengkang. Reasonable colon cancer radical surgery Resection range. Foreign Medical Surgery Volume, 1989, (1): 9-10).

The high-risk factors of colon cancer include the following aspects. 1. Dietary factors: Insufficient intake of trace elements such as high-fat and high-protein diet, lack of vegetables, fruits, cereals, cellulose, calcium and selenium (Terry P, Giovannuci E. Michels KB, et al. Fruit, Vegetables, Dietary fiber, and Risk of Colorectal Cancer.Journal of the National Cancer Institute, 2001, 93: 525). 2. Disease factors: history of lower gastrointestinal disease, cholecystectomy, appendicitis, hypertriglyceridemia (Masafumi T, Joji K, Hirokazu N. Hypertriglyceridemia is Positively Correlated with the Development of Colorectal Tubular Adenoma Adenoma In Japanese Japanese Men.World Journal) Gastroenterology, 2006, 12 (8): 1261-1264) and so on. 3. Lifestyle and other factors: obesity and lack of physical exercise, constipation, genetics, etc.

Colon cancer is treated with surgical resection of the tumor as the first treatment method, supplemented by chemotherapy, radiotherapy, targeted therapy and gene therapy. Colon mesenteric resection is the most important method for surgical treatment of colon cancer, but 25% of colon cancer patients are already in the advanced stage when diagnosed, and because of the biological characteristics of colon cancer with high recurrence and distant metastasis, there are more than 25 % Of patients will still have recurrence or metastasis after the first operation (Wang Guanglin, Meng Zesong, Wang Feifei, etc. Research progress of preoperative chemotherapy for locally advanced colon cancer. Tumor, 2018, 38: 716-722). And surgical treatment is only suitable for some or early patients, because a large number of patients already have more malignant unresectable metastases, so choose systemic treatment (chemotherapy and targeted therapy) or may prolong the overall survival of patients.

The internationally recognized first-line plan for colon cancer chemotherapy is 5-fluorouracil combined with oxaliplatin, or capecitabine combined with oxaliplatin (Andre T, Boni C, Mounedji, L, et al. Oxaliplatin, Fluorouracil, and Leucovorin). Adjuvant Treatment for Colon.Cancer.New England Journal of Medicine, 2004,350 (23): 2343-2351; Schmoll HJ, TaberneroJ, MarounJ, et.al.CapecitabineplusOxaliplatinCompared withFluorouracil / Folinic Colon: Cancer: Final Results of the NO16968 Randomized Controlled Phase III Trial. Journal of Clinical Oncology, 2015, 33 (32): 3733-3740). Multi-drug resistance in cancer chemotherapy is the main reason for the failure of chemotherapy. Colon cancerous tissues are due to carrier proteins related to anti-cancer drug resistance (such as P glycoprotein, multi-drug resistance-related protein or breast cancer resistance protein) Over-expression (Herraez E, Gonzalez SE, Vaquero J, et Al. Cisplatin Induced Chemoresistance in Colon Cancer Cells Involves FXR-Dependent and FXR-Independent Up-regulation of ABC Proteins.Molecular Pharmaceutics (2012): 2576), the anti-cancer drugs in colon cancer cells are rapidly effluxed, the concentration is too low, which leads to the failure of chemotherapy.

Nowadays, targeted therapy of colon cancer has attracted much attention. But colon cancer is a highly heterogeneous and complicated molecular typing disease. At present, traditional clinical targeted therapy mainly targets vascular endothelial growth factor receptor (VEGF) and epidermal growth factor receptor (EGFR), but anti-VEGF drugs have no obvious effect on advanced colon cancer (HurwitzHI, LymanGH.Registries and Randomized Trials Assessing the Effects of Bevacizumab in Colorectal Cancer: Is There There a Common Theme? Journal Journal of Clinical Oncology. 2012, 30 (6): 580-581), and will increase the incidence of arterial vascular events in elderly patients (Ranpura V, Hapani S, Wu S. Treatment-related Mortality with Bevacizumab in Cancer Patients: a Meta-analysis. JAMA, 2011, 305 (5): 487-494); and anti-EGFR drugs are for patients with RAS mutant colon cancer Invalid (Vale CL, Tierney JF, Fisher D, et Al. Does Anti-EGFR Therapy Improve Outcome In Advanced Advanced Colorectal Cancer? A Systematic Review Review and Meta-analysis. Cancer Treatment Reviews, 2012, 38 (6): 618-625) . A genome-wide study of colon cancer shows that the number of tumor gene mutations is very high, with an average of about 75 mutations per tumor, indicating that cancer treatments for specific molecular abnormalities may only be effective in a small proportion of colon cancer patients. At present, there are no biomarkers that can be used in clinical treatment, and only rely on the three types of markers of microsatellite instability (MSI), RAS mutation and BRAF mutation to evaluate clinical diagnosis and treatment decisions or judge the prognosis of treatment (Guinney J, Dienstmann R, Wang X, et Al. The Consensus Molecular Subtypes of Colorectal Cancer. Nat At Med. 2015, 21: 1350-1356; Hutchins G1, Southward K, Handley K, et Al. Value Mismatch Repair, KRAS, and BRAF Mutations in Predicting Recurrence and Benefits from Chemotherapy in Colorectal Cancer. Journal of Clinical Oncology. 2011, 29: 1261-1270).

Since the incidence and mortality of colon cancer are at a high level worldwide, it has seriously endangered people's health and caused a heavy burden on families and society. However, colon cancer has high recurrence, distant metastasis and high heterogeneity Biological biological characteristics, so the current clinical treatments are very scarce and can not significantly increase the clinical benefit of colon cancer patients, there is an urgent need to develop new treatments or drugs to cope with the increasing status of colon cancer morbidity and mortality .

Selenium is one of the essential trace elements in the human body. The reduction of selenium in the blood will induce a variety of diseases including tumors and cardiovascular diseases (Reeves MA, Hoffmann PR. The Human Selenoproteome: Recent Insights Functions to Regulations. Cellular and Molecular Life Sciences, 2009, 66 (15): 2457-78). Because of its application in anti-tumor, anti-virus and treatment of nervous system-related diseases, selenium-containing drugs have become a hot spot for domestic and foreign scholars. Drug research mainly focuses on anti-tumor, anti-inflammatory and anti-hypertension (RomualdoC , Stefania C, Marina DG, et al. Novel Selenium-containing Non-natural Diamino acids. Tetrahedron Letters, 2007, 48 (7): 1425-1427).

Malcolm FGStevens and other US patents (Malcolm FGStevens, Andrew D. Westwell, Mei-Sze Chua, et al. Substituted 2-arylbenzazole compounds and the use of them) USitus agents (US6858633B1) published the following formula (II) It also pointed out the use of these compounds in cancer, but the content of the description is mainly for breast cancer, because the pathogenesis and treatment pathways between different cancers are basically different, and most anti-cancer compounds are only sensitive to certain Cancer has a therapeutic effect, so whether this type of compound works in other cancers is still unknown.

The inventor of this patent, Shi Dongfang, has published articles on the synthesis of benzothiazole compounds and their anti-breast cancer effects in vivo and in vitro (Dong-Fang Shi, Tracey D. Bradshaw, Samantha Wrigley, et al. Antitumor Benzothiazoles. of 2- (4-Aminophenyl) benzothiazoles and Evaluation of Their Activities against Breast Cancer Cell Lines in Vitro and in Vivo. Journal of Medicinal Chemistry, 1996, 39: 3375-3384), meanwhile, the present inventor Shi Dongfang applied for Chinese patents (Shi Dongfang, Fu Changjin, Cheng Xi, etc. Compounds for the treatment or prevention of breast cancer. CN201610299350.1) published the benzoselenazole-2-benzene compounds shown in formula (III) and their anti-breast cancer Pharmacological activity. These two compounds were human breast cancer cells nanomolar inhibitory effect, wherein the benzothiazole compound of ER ⁺ (MCF-7 cell lines, and BO) and ER ^- (MT-1 and MT-3 cell line) The breast cancer xenografts of nude mice all show very significant tumor suppressive effects, but these two types of compounds have no inhibitory activity against other tumor cell lines such as prostate cancer, bladder cancer, melanoma, lung cancer, liver cancer, and esophageal cancer.

So far, there is no report on the effect of 4- (benzoselazol-2-yl) arylamine compounds on gastric or intestinal cancer models.

Summary of the invention

The purpose of the present invention is to provide a class of 4- (benzoselazol-2-yl) aromatic amine compounds for the treatment of gastric cancer or intestinal cancer based on the prior art.

The object of the present invention can be achieved by the following measures:

The use of a compound represented by general formula (IV) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating or preventing gastric cancer or intestinal cancer,

Further, the use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating or preventing gastric cancer or intestinal cancer,

In each formula,

R ¹ and R ² are independently selected from H, D, halogen, -CN, C _1-3 alkyl, substituted C _1-3 alkyl, C _1-3 alkoxy or substituted C _1-3 alkyl Oxy;

R ^{3 is} selected from H, halogen, -OH, -CN, -C (= O) NH ₂ , substituted -C (= O) NH ₂ , C _1-3 alkyl, substituted C _1-3 alkyl, C _2-3 alkenyl, C _2-3 alkynyl, C _1-3 alkoxy or substituted C _1-3 alkoxy;

R ^{4 is} selected from H, D, halogen, -OH, -CN, -NH ₂ , substituted -NH ₂ , -C (= O) NH ₂ , substituted -C (= O) NH ₂ , C _1-3 One or both of alkyl, substituted C _1-3 alkyl, C _2-3 alkenyl, C _2-3 alkynyl, C _1-3 alkoxy, or substituted C _1-3 alkoxy ;

n = 1 or 2;

R ^{5 is} selected from H, -CN, -OH, C _1-3 alkyl, C _1-3 alkoxy or amino acid residues;

Z is selected from CH or N;

The substituents in R ¹ , R ² , R ³ or R ⁴ are selected from D, halogen, OH, C _1-3 alkyl or C _1-3 alkoxy.

In a preferred embodiment, R ¹ and R ² in the present invention are independently selected from H, D, halogen, -CN, C _1-3 alkyl, substituted C _1-3 alkyl, C _1-3 Alkoxy or substituted C _1-3 alkoxy, the substituent is selected from D, F or C _1-3 alkoxy;

In another preferred embodiment, R ¹ and R ² in the present invention are independently selected from H, D, F, Cl, —CN, —CH ₃ , —CF ₃ , —OCF ₃ or —OCHF ₂ .

In another preferred embodiment, R ¹ in the present invention is selected from D, F, Cl, -CN, -CH ₃ or -CF ₃ .

In another preferred embodiment, R ² in the present invention is selected from H.

In a preferred embodiment, R ³ in the present invention is selected from H, halogen, -CN, -C (= O) NH ₂ , C _1-2 alkyl, C _2-3 alkenyl, C _2-3 alkyne Group, C _1-3 alkoxy or substituted C _1-3 alkoxy.

In another preferred embodiment, R ³ in the present invention is selected from H, F, Cl, Br, —CN, —C (═O) NH ₂ , —CH ₃ , —CH ₂ CH ₃ , —CF ₃ , -CH = CH ₂ , -C≡CH, -OCHF ₂ or -OCF ₃ .

In a preferred embodiment, R ⁴ in the present invention is selected from H, D, halogen, -CN, C _1-3 alkyl, substituted C _1-3 alkyl, C _1-3 alkoxy or substituted One or two of C _1-3 alkoxy groups.

In another preferred embodiment, R ⁴ in the present invention is selected from H, D, F, Cl, Br, I, -CN, -CH ₃ , -CF ₃ , -OCH ₃ , -OCH ₂ CH ₃ ,- OCHF ₂ or -OCF ₃ .

In a preferred embodiment, R ⁵ in the present invention is selected from H, C _1-3 alkyl, C _1-3 alkoxy or amino acid residues.

In another preferred embodiment, R ⁵ in the present invention is selected from H, —CH ₃ , —CF ₃ , —OCH ₃ or 2,6-diamino-hexanoyl.

In a preferred embodiment, the compound involved in the present invention is selected from:

2-amino-5- (5-fluorobenzoselenazol-2-yl) benzonitrile,

2-amino-3-fluoro-5- (5-fluorobenzoselenazol-2-yl) benzonitrile,

2-ethynyl-4- (5-fluorobenzoselenazol-2-yl) aniline,

5-fluoro-4- (5-fluorobenzoselenazol-2-yl) -2-methylaniline,

2-bromo-6- (5-fluorobenzoselenazol-2-yl) pyridine-3-amine,

3-amino-6- (5-fluorobenzoselenazol-2-yl) -2-carboxamidopyridine,

4- (5-fluorobenzoselenazol-2-yl) -2-methylaniline,

2-bromo-4- (5-fluorobenzoselenazol-2-yl) -6-methylaniline,

2-bromo-6-fluoro-4- (5-fluorobenzoselenazol-2-yl) aniline,

2,6-difluoro-4- (5-fluorobenzoselenazol-2-yl) aniline,

2-fluoro-4- (5-fluorobenzoselenazol-2-yl) -6-methylaniline.

Unless otherwise specified, each group involved in the present invention has the following meanings.

"H", or hydrogen, refers to protium (1H), which is the main stable isotope of hydrogen.

"D", deuterium, refers to a stable form of hydrogen isotope, also known as heavy hydrogen, and its element symbol is D.

"Halogen" means fluorine atom, chlorine atom, bromine atom or iodine atom.

"-OH" refers to a hydroxyl group.

"-NH ₂ " refers to an amino group.

"-CONH ₂ ", ie C (= O) -NH ₂ , refers to an amide group.

"-CN" means a cyano group.

"-NO ₂ " refers to a nitro group.

"Alkyl" refers to a saturated aliphatic hydrocarbon group of 1-10 carbon atoms, including straight-chain and branched-chain groups (the numerical range mentioned in this application, for example "1-10", refers to this group In this case, it is an alkyl group, which may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 10 carbon atoms). An alkyl group containing 1-4 carbon atoms is called a lower alkyl group. When the lower alkyl group has no substituent, it is called an unsubstituted lower alkyl group. The alkyl can be selected from C _1-6 alkyl, C _1-5 alkyl, C _1-4 alkyl, C _1-3 alkyl, C _1-2 alkyl, C _2-3 alkyl, C _2-4 Alkyl and so on. Specific alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl, and the like. The alkyl group may be substituted or unsubstituted.

"Alkenyl" means an unsaturated hydrocarbon group having at least one carbon-carbon double bond, including straight-chain and branched-chain groups (the numerical range mentioned in this application, such as "2-5", refers to this group At this time, it is an alkenyl group, which may contain 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to 5 carbon atoms). The alkenyl in the present invention may be C _2-8 alkenyl, C _2-6 alkenyl, C _2-5 alkenyl, C _2-4 alkenyl, C _2-3 alkenyl, etc. Specific alkenyl groups include but It is not limited to vinyl, propenyl, and butenyl.

"Alkynyl" means an unsaturated hydrocarbon group having at least one carbon-carbon triple bond, including straight-chain and branched-chain groups (the numerical range mentioned in this application, such as "2-5", refers to this group In this case, it is an alkynyl group, which may contain 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to 5 carbon atoms). The alkynyl group in the present invention may be C _2-8 alkynyl, C _2-6 alkynyl, C _2-5 alkynyl, C _2-4 alkynyl, C _2-3 alkynyl, etc. Specific alkenyl groups include but It is not limited to ethynyl, propynyl and butynyl.

"Alkoxy" means -O- (unsubstituted alkyl) and -O- (unsubstituted cycloalkyl) groups, which further means -O- (unsubstituted alkyl). Representative embodiments include but are not limited to methoxy, ethoxy, propoxy, cyclopropoxy, and the like.

"Amino acid residue" refers to a group formed by an amino acid lacking a certain group (such as -OH, -COOH, or -NH ₂ ), where amino acids include, but are not limited to, 20 naturally occurring species usually designated by three-letter symbols Amino acids, and also includes β-alanine, citrulline, demosine, γ-aminobutyric acid, homocysteine, homoserine, 4-hydroxyproline, hydroxylysine, iso Isodemosine, 3-methylhistidine, norvaline, methioninesulfone and ornithine. An example of an amino acid residue includes, but is not limited to: 2,6-diamino-hexanoyl.

"Pharmaceutically acceptable salt" is a salt comprising a compound of general formula (I) formed with an organic acid or an inorganic acid, and means those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) Forming a salt with an acid, obtained by the reaction of the free base of the parent compound with an inorganic acid or an organic acid, such as (but not limited to) hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid And perchloric acid, organic acids such as (but not limited to) acetic acid, propionic acid, acrylic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, hydroxybenzoic acid, γ-hydroxybutyric acid , Methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, Mandelic acid, succinic acid or malonic acid etc.

(2) The salts of acid protons present in the parent compound are replaced by metal ions or coordinated with organic bases, metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions, organic bases such as ethanolamine, diethanolamine, trivalent Ethanolamine, tromethamine, N-methylglucamine, etc.

"Pharmaceutical composition" refers to a mixture of one or more compounds described herein or their pharmaceutically acceptable salts and prodrugs with other chemical ingredients, such as pharmaceutically acceptable carriers and excipients . The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.

In the application of the compound of the present invention, the compound of the present invention, a pharmaceutically acceptable salt or a solvate thereof can be used as an active ingredient or a main active ingredient, supplemented with a pharmaceutically acceptable auxiliary material to make a pharmaceutical composition, and then The patient is administered.

The compounds of the present invention can be formulated into any clinically or pharmaceutically acceptable dosage form in a manner known in the art. For oral administration, it can be made into conventional solid preparations, such as tablets, capsules, pills, granules, etc .; or oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When preparing an oral preparation, a suitable filler, binder, disintegrant, lubricant, etc. may be added. When used for parenteral administration, injections can be prepared, including injections, sterile powders for injection and concentrated solutions for injection. When it is prepared as an injection, it can be produced by a conventional method in the existing pharmaceutical field. When preparing an injection, an additional agent may not be added, or an appropriate additional agent may be added according to the nature of the medicine.

The present invention also provides a method for treating human gastric cancer or intestinal cancer, that is, administering a compound of the present invention, a pharmaceutically acceptable salt, or a solvate thereof to a person suffering from gastric cancer or intestinal cancer at 0.1-1000 mg per dose Drug, or the pharmaceutical composition of the present invention is administered at 0.1-1000 mg per dose.

The 4- (benzoselenazol-2-yl) arylamine compounds involved in this patent have anti-breast cancer pharmacological activity, but in vitro tests have shown that for prostate cancer, bladder cancer, melanoma, lung cancer, liver cancer, Tumor cell lines such as esophageal cancer have no obvious inhibitory effect. However, this patent found that these compounds have excellent therapeutic effects on gastric cancer or intestinal cancer: they have a significant growth inhibition effect on human gastric cancer cell lines MKN-45, AGS and NCI-N87, and the effect is basically close to the positive control drug paclitaxel ; These compounds also show excellent tumor growth inhibition effect on human gastric cancer MKN-45 and AGS xenograft tumors in nude mice, and the tumor inhibition rate is greater than 35%; so these compounds can be used in the treatment of human gastric cancer. This patent discovered that such compounds have a significant growth inhibition effect on human colon cancer cell lines HCT116, HT29, HCT15 and COLO205, and the effect is basically close to that of the positive control drug paclitaxel; Xenograft tumors also exhibit excellent tumor growth inhibition effects, with tumor inhibition rates greater than 40%. Therefore, these compounds can be used in the treatment of human bowel cancer.

The present invention finds a new use of 4- (benzoselenazol-2-yl) arylamine compounds in the treatment of diseases and provides a potential drug for the treatment of gastric cancer or intestinal cancer.

BRIEF DESCRIPTION

Figure 1 is the inhibitory effect of compound 6 on the tumor volume of human gastric cancer cell line AGS xenograft in nude mice;

Figure 2 is the inhibitory effect of compound 6 on the tumor volume of human gastric cancer cell line MKN-45 xenograft in nude mice;

Figure 3 is the inhibitory effect of compound 22 on the tumor volume of human gastric cancer cell line AGS xenograft in nude mice;

4 is the inhibitory effect of compound 22 on the tumor volume of human gastric cancer cell line MKN-45 xenograft in nude mice;

Figure 5 is the inhibitory effect of compound 23 on the tumor volume of human gastric cancer cell line AGS xenograft in nude mice;

Figure 6 is the inhibitory effect of compound 23 on the tumor volume of human gastric cancer cell line MKN-45 xenograft in nude mice;

7 is the inhibitory effect of compound 6 on the tumor volume of human colon cancer cell line HT29 xenograft in nude mice;

8 is the inhibitory effect of compound 6 on the tumor volume of human colon cancer cell line HCT116 xenograft in nude mice;

Figure 9 is the inhibitory effect of compound 22 on the tumor volume of human colon cancer cell line HT29 xenograft in nude mice;

Figure 10 is the inhibitory effect of compound 22 on the tumor volume of human colon cancer cell line HCT116 xenograft in nude mice;

Figure 11 is the inhibitory effect of compound 23 on the tumor volume of human colon cancer cell line HT29 xenograft in nude mice;

Fig. 12 shows the inhibitory effect of compound 23 on the tumor volume of human colon cancer cell line HCT116 xenograft in nude mice.

detailed description

The content of the present invention will be further described below in conjunction with embodiments, but the scope of protection of this patent is not limited to the following embodiments.

Example 1: Synthesis of 2-amino-5- (5-fluorobenzoselenazol-2-yl) benzonitrile (6)

Step A: Add 2-nitro-4-fluoroaniline (5.0g, 32.0mmol) in methylene chloride (80mL) dropwise to boron trifluoride etherate (6.82g, 48.1mmol) at -20 ～ -25 ° C )in. After stirring for 15 minutes, a solution of isoamyl nitrite (4.50 g, 38.4 mmol) in dichloromethane (20 mL) was added dropwise at this temperature. After the addition is complete, stirring is continued for 30 minutes, and then at -10 to 0 ° C for 30 minutes. To the reaction system was added dropwise cooled petroleum ether (60 mL), filtered, and the filter cake was washed with cooled methyl tert-butyl ether (10 mL) to obtain 2-nitro-4-fluorophenyl-fluoroboronic acid diazonium salt ( 1) Crude product (8.10g). This compound was directly used in the next reaction without purification.

Step B: Under an ice water bath, to a mixture of crude compound 1 (8.10 g) and water (150 mL) was added dropwise a solution of potassium selenocyanate (4.35 g, 30.2 mmol) in water (20 mL). After the addition was complete, stirring was continued for 1 hour. Filter and dissolve the filter cake with dichloromethane (150 mL). The insoluble matter was removed by filtration, and the filtrate was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain crude 4-fluoro-2-nitro-1-selenocyanate (2) (6.10 g). This compound was directly used in the next reaction without purification.

Step C: Sodium metal (3.23 g, 140 mmol) was added to a mixture of crude compound 2 (6.10 g) and absolute ethanol (90 mL) at room temperature, and the resulting mixture was stirred for 1 hour under a water bath. Cooled to 0 ~ 5 ℃, filtered, the filter cake was washed with a small amount of cooled ethanol, to obtain 1,2-bis (4-fluoro-2-nitrophenyl)-diselenide (3) crude product (3.90g). This compound was directly used in the next reaction without purification.

Step D: The crude compound 3 (3.90 g) was dissolved in ethanol (60 mL), stannous chloride (7.90 g, 41.7 mmol) was added, and the resulting mixture was stirred under reflux under nitrogen for 4 hours. Most of the solvent was distilled off under reduced pressure, water (120 mL) and ethyl acetate (200 mL) were added, and the pH value was adjusted to 8-9 with 2M sodium hydroxide solution. The insoluble material was removed by filtration through Celite, the layers were separated, the aqueous layer was extracted with ethyl acetate (60 mL × 2), and the combined organic layer was dried over anhydrous sodium sulfate. Then it was purified by reduced pressure column chromatography (200-300 mesh silica gel, ethyl acetate elution), and the resulting product was recrystallized from petroleum ether to obtain 6,6'-diselenyl-bis (3-fluoroaniline) (4 ) (3.0g). The total yield of the four-step reaction in steps A, B, C and D was 49.6%.

Step E: To a solution of compound 4 (3.0 g, 7.93 mmol) and 4-amino-3-bromobenzoic acid (1.70 g, 7.87 mmol) in toluene (50 mL) was added tributylphosphine (8.0 g, 39.5 mmol), The resulting mixture was stirred at reflux under nitrogen for 48 hours. Cool to room temperature, add water (50 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL × 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether = 1:50 ~ 1: 5 elution) to give 2-bromo-4- (5-fluorobenzo Selenazol-2-yl) aniline (5) (1.60g). The yield was 54.9%.

Step F: A mixture containing compound 5 (400 mg, 1.08 mmol), cuprous cyanide (145 mg, 1.62 mmol) and NMP (10 mL) was stirred at 150 ° C. overnight. Cool to room temperature, add water (40 mL), and adjust the pH to 8-9 with 2M sodium carbonate solution. It was extracted with ethyl acetate (30 mL × 3), and the combined organic phase was washed with saturated brine (20 mL × 2) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200 to 300 mesh silica gel, ethyl acetate: dichloromethane: petroleum ether = 1: 1: 20 to 1: 1: 8 elution) to give 2-amino 5- (5-fluorobenzoselenazol-2-yl) benzonitrile (6) (84 mg). The yield was 54.9%. ¹ H NMR (DMSO-d6, 400 MHz) δ 8.15-8.12 (m, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.98-7.96 (m, 1H), 7.79-7.76 (m, 1H) , 7.24-7.19 (m, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.83 (s, 2H). MS (EI, m / z): 316.0 [MH] ^- .

Example 2: Synthesis of 2-amino-3-fluoro-5- (5-fluorobenzoselenazol-2-yl) benzonitrile (9)

Step A: To a solution of compound 4 (4.30 g, 11.4 mmol) and 4-amino-3-fluorobenzoic acid (1.76 g, 11.3 mmol) in toluene (50 mL) was added tributylphosphine (11.5 g, 39.5 mmol), The resulting mixture was stirred at reflux under nitrogen for 48 hours. Cool to room temperature, add water (50 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL × 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether = 1: 100 ~ 1:20 elution) to give 2-fluoro-4- (5-fluorobenzo Selenazol-2-yl) aniline (7) (780 mg). The yield was 22.3%.

Step B: NBS (585 mg, 3.29 mmol) was added to a solution of compound 7 (780 mg, 2.52 mmol) in DMF (10 mL). After the addition, the resulting mixture was stirred at room temperature for 30 minutes. Water (40 mL) was added and extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with water (15 mL), saturated aqueous sodium bicarbonate solution (15 mL) and saturated brine (15 mL) in this order, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was recrystallized from petroleum ether / ethyl acetate to obtain 2-bromo-6-fluoro-4- (5-fluorobenzoselenazol-2-yl) aniline (8) (720 mg). The yield was 73.6%. ¹ H NMR (DMSO-d ₆ , 500 MHz) δ 8.16-8.13 (m, 1H), 7.90 (s, 1H), 7.79 (dd, J = 2.0, 10.0 Hz, 1H), 7.73 (dd, J = 2.0 , 10.0 Hz, 1H), 7.24-7.20 (m, 1H), 6.12 (s, 2H). MS (EI, m / z): 388.9 [M + H] ⁺ .

Step C: A mixture containing compound 8 (520 mg, 1.34 mmol), cuprous cyanide (180 mg, 2.01 mmol) and NMP (10 mL) was stirred at 150 ° C overnight. Cool to room temperature, add water (40 mL), and adjust the pH to 8-9 with 2M sodium carbonate solution. It was extracted with ethyl acetate (30 mL × 3), and the combined organic phase was washed with saturated brine (20 mL × 2) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether = 1:50 ~ 1:20 elution) to give 2-amino-3-fluoro-5- (5 -Fluorobenzoselenazol-2-yl) benzonitrile (9) (150 mg). The yield was 33.5%. ¹ H NMR (DMSO-d6, 400 MHz) δ 8.19-8.16 (m, 1H), 7.99-7.94 (m, 2H), 7.80 (dd, J = 2.4, 10.0 Hz, 1H), 7.27-7.23 (m, 1H), 6.99 (s, 2H). MS (EI, m / z): 336.0 [M + H] ⁺ .

Example 3: Synthesis of 2-ethynyl-4- (5-fluorobenzoselenazol-2-yl) aniline (12)

Step A: A mixture containing compound 5 (500 mg, 1.35 mmol), acetic anhydride (0.5 mL), pyridine (10 mL) and 4-dimethylaminopyridine (10 mg, 0.0819 mmol) was stirred at 90 ° C overnight. After cooling to room temperature, water (50 mL) was added and extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with saturated brine (15 mL × 2) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: dichloromethane: petroleum ether = 1: 1: 25 ～ 1: 1: 10 elution) to obtain N- [2 -Bromo-4- (5-fluorobenzoselenazol-2-yl) phenyl] acetamide (10) (500 mg). The yield was 89.8%.

Step B: Add trimethylsilane acetylene (131 mg, 1.33 mmol) to the compound containing compound 10 (500 mg, 1.21 mmol), bistriphenylphosphine palladium dichloride (40 mg, 0.0570 mmol), triethylamine (40 mL) via syringe ) And DMF (4 mL), after the addition, the resulting mixture was stirred at 50 ° C. overnight. Most of the solvent was distilled off under reduced pressure, water (30 mL) was added, and extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with saturated brine (15 mL × 2) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether = 1:50 ~ 1:20 elution) to obtain N- {4- (5-fluorobenzoselenide Oxazol-2-yl) -2-[(trimethylsilyl) ethynyl] phenyl} acetamide (11) (211 mg). The yield was 40.6%.

Step C: A mixture containing compound 11 (100 mg, 0.233 mmol), 2M sodium hydroxide solution (10 mL), THF (5 mL) and methanol (10 mL) was stirred at 80 ° C for 1 hour. After cooling to room temperature, water (30 mL) was added and extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with saturated brine (15 mL × 2) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting product was recrystallized from petroleum ether / ethyl acetate to obtain 2-ethynyl-4- (5-fluorobenzoselenazol-2-yl) aniline (12). ¹ H NMR (DMSO-d6, 400 MHz) δ 8.13-8.09 (m, 1H), 7.82 (d, J = 2.0 Hz, 1H), 7.77-7.73 (m, 2H), 7.22-7.19 (m, 1H) , 6.81 (d, J = 8.4 Hz, 1H), 6.20 (s, 2H), 4.48 (s, 1H). MS (EI, m / z): 315.0 [MH] ^- .

Example 4: Synthesis of 5-fluoro-4- (5-fluorobenzoselenazol-2-yl) -2-methylaniline (16)

Step A: In an ice water bath, add NBS (3.55g, 19.9mmol) in portions to a solution of 5-fluoro-2-methylaniline (2.50g, 20.0mmol) in DMF (20mL). After the addition is complete, the result is The mixture continued to stir at this temperature for 1 hour. Water (80 mL) was added and extracted with ethyl acetate (60 mL × 3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (30 mL × 2) and saturated brine (30 mL × 2), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain crude 4-bromo-3-fluoro-6-methylaniline (13) (4.87g). This compound was directly used in the next reaction without purification.

Step B: A mixture containing crude compound 13 (4.87 g), cuprous cyanide (2.63 g, 29.4 mmol) and NMP (15 mL) was stirred at 180 ° C. under nitrogen for 7 hours. Water (75 mL) was added and extracted with ethyl acetate (50 mL × 3). The combined organic phase was washed with water (30 mL × 2) and saturated brine (30 mL) in this order, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether = 1:30 ~ 1: 5 elution) to give 4-amino-2-fluoro-5-methyl Benzonitrile (14) (1.74g). The total yield of the two-step reaction in steps A and B is 58.2%.

Step C: A mixture containing compound 14 (1.74 g, 11.6 mmol), 1M sodium hydroxide solution (50 mL) and ethanol (5 mL) was stirred at reflux overnight. Cool to room temperature, add water (50 mL), extract with methyl tert-butyl ether (20 mL x 2), and the product is in the aqueous phase. The aqueous phase was adjusted to pH 3-4 with 2M hydrochloric acid, and solid precipitated. Filter and dry the filter cake to give 4-amino-2-fluoro-5-methylbenzoic acid (15) (1.75g). The yield was 89.2%.

Step D: To a solution of compound 4 (1.0 g, 2.64 mmol) and compound 15 (447 mg, 2.64 mmol) in toluene (30 mL) was added tributylphosphine (2.68 g, 13.2 mmol), and the resulting mixture was stirred under reflux under nitrogen 36 hour. Cool to room temperature, add water (40 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL × 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: petroleum ether = 1:50 ~ 1:10 elution) to obtain 5-fluoro-4- (5-fluorobenzo Selenazol-2-yl) -2-methylaniline (16) (655 mg). The yield was 72.9%. ¹ H NMR (DMSO-d6, 400 MHz) δ 8.14-8.10 (m, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.78 (dd, J = 2.4, 10.0 Hz, 1H), 7.22-7.17 (m, 1H), 6.54-6.51 (m, 1H), 6.08 (s, 2H), 2.13 (s, 3H). MS (EI, m / z): 323.0 [MH] ^- .

Example 5: Synthesis of 2-bromo-6- (5-fluorobenzoselenazol-2-yl) pyridine-3-amine (18)

Step A: To a solution of compound 4 (1.80 g, 4.76 mmol) and 5-aminopyridine-2-carboxylic acid (657 mg, 4.76 mmol) in toluene (50 mL) was added tributylphosphine (4.82 g, 23.8 mmol), and the resulting mixture Stir under reflux for 36 hours under nitrogen. Cool to room temperature, add water (40 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL x 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate: dichloromethane: petroleum ether = 1: 1: 20 ~ 1: 1: 5 elution) to give 6- (5 -Fluorobenzoselenazol-2-yl) pyridine-3-amine (17) (712 mg). The yield was 51.2%.

Step B: Under ice-water bath, NBS (61 mg, 0.343 mmol) was added portionwise to a solution of compound 17 (100 mg, 0.342 mmol) in dichloromethane (6 mL). After the addition was complete, the resulting mixture continued to be stirred at this temperature 0.5 hours. Water (15 mL) was added and extracted with dichloromethane (60 mL × 3). The combined organic phase was washed with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL) in this order, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, THF: dichloromethane: petroleum ether = 1: 1: 20 ~ 1: 1: 10 elution) to give 2-bromo-6- (5-fluorobenzoselenazol-2-yl) pyridine-3-amine (18). ¹ H NMR (DMSO-d6, 400 MHz) δ 8.15-8.11 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.80 (dd, J = 2.4, 10.0 Hz, 1H), 7.26-7.19 (m, 2H), 6.37 (s, 2H). MS (EI, m / z): 369.9 [MH] ^- .

Example 6: Synthesis of 3-amino-6- (5-fluorobenzoselenazol-2-yl) -2-carboxamidopyridine (19)

Using compound 18 as the starting material, the experimental operation for synthesizing compound 19 was prepared according to the method of step F in Example 1 (since this batch of compound 18 was not purified by column chromatography, there was a little sodium bicarbonate and water in the post-treatment It was so clean that compound 19) was formed in this reaction. ¹ H NMR (DMSO-d6, 400 MHz) δ 8.16-8.12 (m, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.82 (dd, J = 2.4, 10.0 Hz, 1H), 7.76 (s , 1H), 7.65 (s, 1H), 7.54 (s, 2H), 7.30 (d, J = 8.8 Hz, 1H), 7.26-7.21 (m, 1H). MS (EI, m / z): 335.0 [MH] ^- .

Example 7:

Compound 4- (5-fluorobenzoselenazol-2-yl) -2-methylaniline (20), 2-bromo-4- (5-fluorobenzoselenazol-2-yl) -6-methyl Aniline (21), 2-bromo-6-fluoro-4- (5-fluorobenzoselenazol-2-yl) aniline (22), 2,6-difluoro-4- (5-fluorobenzoselenazole For the synthesis of 2-yl) aniline (23) and 2-fluoro-4- (5-fluorobenzoselenazol-2-yl) -6-methylaniline (24), see patent CN201610299350.1 or US10005744B2.

Example 8: Compounds inhibit growth of human gastric cancer cell lines AGS, MKN-45 and NCI-N87

1. Name and source of experimental materials

Human gastric cancer cell lines AGS, MKN-45 and NCI-N87 were purchased from the Cell Resource Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences. Paclitaxel, Resazurin and methylene blue were purchased from Sigma-Aldrich Co., LLC; potassium ferricyanide and potassium ferrocyanide were purchased from Aladdin Reagent Co., Ltd .; DMEM medium, 1640 medium, phenol red-free DMEM and fetal bovine serum Purchased from Thermo Fisher Inc .; Penicillin and Streptomycin were purchased from Biyuntian Biotechnology Co., Ltd.

2. Experimental methods

DMEM medium for AGS (containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin), MKN-45 1640 medium (containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin), NCI-N87 with DMEM medium (containing 20% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin), three cells were cultured at 37 ℃, 5% CO ₂ incubator To the cell density of about 90%.

The cells were seeded in 96-well plates at 3 × 10 ³ / well, and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours.

Prepare different concentrations of test compound or control drug paclitaxel with medium, and add 100μL / well to 96-well plate as test compound well or control drug well; add 100μL / well medium without test compound or control drug As a negative control well. Incubate at 37 ° C, 5% CO ₂ , AGS and MKN-45 cells were cultured for 72 hours, and NCI-N87 cells were cultured for 120 hours.

Dissolve Resazurin (15mg / 50mL, 200 ×), methylene blue (25mg / 10mL, 1000 ×), potassium ferricyanide (0.329g / 100mL, 100 ×) and potassium ferrocyanide (0.422g / 100mL, 100 ×) Prepare 10 × Alamar Blue solution in PBS (0.1M, pH = 7.4), then dilute with phenol red-free DMEM medium to 1 × Alamar Blue solution, and prepare immediately before use.

The cells in the 96-well plate were carefully washed twice with PBS (0.1M, pH = 7.4), and the PBS was exhausted, and then added 1 × Alamar Blue solution at 100 μL / well; 100 μL 1 × Alamar Blue was added to the cell-free wells The solution serves as a blank control well. Incubate the 96-well plate at 37 ° C in a 5% CO ₂ incubator for 3 hours.

The fluorescence value of the cells was detected at 530nm at Ex 530 / Em with Victor X4 (Perkin Elmer). The fluorescence value of the test compound well is represented by F _{(test compound)} ; the fluorescence value of the blank control well is represented by F _{(blank control)} ; the fluorescence value of the negative control well is represented by F _{(negative control)} . Calculate the cell survival rate under different drug concentrations according to the following formula. Set 3 replicate wells at each concentration to obtain the average and standard deviation.

Prism Graph software was used to calculate the half-inhibitory concentration (IC ₅₀ ) of the test compound or control drug on the cells according to the cell survival rate.

3. Experimental results

The test results are shown in Table 1. The compounds have a significant inhibitory effect on the growth of human gastric cancer cell lines AGS, MKN-45 and NCI-N87. Among them, compounds 6, 9, 12, 19, 22 and 23 had better inhibitory effects.

Table 1. Test compound's half-inhibitory concentration of human gastric cancer cell lines AGS, MKN-45 and NCI-N87 (IC ₅₀ , nM)

Example 9: Growth inhibitory activity of compounds 6, 22 and 23 on human gastric cancer AGS and MKN-45 xenograft tumors in nude mice

1. Test materials

1. Experimental animals

SPF grade BALB / c nude mice, female, are 6-8 weeks old at the start of administration and weigh 18-20 g. Provided by Changzhou Cavens Experimental Animal Co., Ltd. (Experimental Animal Production License: SCXK (Su) 2016-0010; Experimental Animal Use License: SYXK (Su) 2017-0007). Purchased animal batch number: 201820473.

2. Test reagent

Polyethylene glycol 400 (PEG400), batch number 20180412, purchased from Chengdu Kelong Chemical Reagent Factory; sodium chloride injection (physiological saline), batch number A17111105, purchased from Hebei Tiancheng Pharmaceutical Co., Ltd .; DMSO, batch number Q6949, purchased from MP Biomedicals.

3. Cell lines

Human gastric cancer AGS and MKN-45 cells were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; AGS was cultured in 1640 medium with 10% fetal bovine serum, and MKN-45 was cultured in DMEM medium with 10% fetal bovine serum to cultivate.

2. Test method

1. Test compound

Low-dose group (administration dose: 2.5 mg / kg): accurately weigh 2.0 mg of the test compound before administration and dissolve it into 8 mL of clear solution with a solvent for intravenous injection (PEG400 25%, DMSO 2.5%, physiological saline 72.5%) Solution, the final concentration of the intravenous solution of the test compound in the low-dose group was 0.25 mg / mL, and the intravenous administration volume was 0.2 mL / 20 g body weight.

High-dose group (administration dose: 5.0 mg / kg): accurately weigh 4.0 mg of the test compound before administration and dissolve it to 8 mL of clear solution with a vehicle for intravenous injection (PEG400 50%, DMSO 5%, saline 45%) Solution, the final concentration of the intravenous injection solution of the test compound in the high-dose group was 0.50 mg / mL, and the intravenous administration volume was 0.2 mL / 20 g body weight.

2. Trial grouping and administration method

The AGS and MKN-45 cells in the logarithmic growth phase were taken and inoculated under the aseptic conditions under the skin of the right armpit of 60 nude mice, respectively. The cell inoculation volume was 5 × 10 ⁶ cells / cell. Use a vernier caliper to measure the diameter of the transplanted tumor. When the tumor grows to about 100mm ³ , 56 AGS and MKN-45 tumor-bearing nude mice with good growth status and good tumor size uniformity are selected and randomly divided into 7 groups, each group 8 Only, the model group, compound 6, 22 and 23 low-dose group and high-dose group. Each group was administered intravenously, and were continuously administered once a day on days 0, 1, 2, 3, and 4 for a total of 5 days. The model group was given an equal volume of vehicle control. Using the method of measuring tumor diameter, the anti-tumor effect of the test compound was dynamically observed. The tumor diameter was measured every two days and weighed at the same time. On the 20th day, the nude mice were sacrificed and the tumor masses were removed and weighed accurately.

The calculation formula of tumor volume (TV) is:

Where a and b denote length and width, respectively.

The formula for calculating the% of tumor suppression is:

3. Test results

The test results are shown in Table 2, Table 3, and Figures 1 to 6. The test compounds 6, 22, and 23 were injected at a dose of 2.5 mg / kg and 5.0 mg / kg in the tail vein for 5 consecutive days, respectively. Significantly inhibit the tumor growth of gastric cancer cells AGS and MKN-45 xenografts in nude mice.

Table 2. Inhibitory effect of compounds on human gastric cancer cell line AGS xenograft tumor growth in nude mice (Mean ± SD, n = 8)

Table 3. Compound inhibitory effect on human gastric cancer cell line MNK-45 xenograft tumor growth in nude mice (Mean ± SD, n = 8)

Example 10: Compounds inhibit growth of human colon cancer cell lines HCT116, HCT15, HT29, SW620 and COLO205

1. Name and source of experimental materials

Human colon cancer cell lines HCT116, HCT15, HT29, SW620 and COLO205 were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Paclitaxel, Resazurin and methylene blue were purchased from Sigma-Aldrich Co., LLC; potassium ferricyanide and potassium ferrocyanide were purchased from Aladdin Reagent Co., Ltd .; DMEM medium, phenol-free DMEM and fetal bovine serum were purchased from Thermo Fisher Scientific Inc .; Penicillin and Streptomycin were purchased from Biyuntian Biotechnology Co., Ltd.

2. Experimental methods

HCT116, HCT15, HT29, SW620 and COLO205 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin) at 37 ° C, 5% CO ₂ incubator to the cell density Up to about 90%.

The cells were seeded in 96-well plates at 3 × 10 ³ / well, and incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours.

The test compound or the control drug paclitaxel with different concentration gradients is prepared with the medium, and added to the 96-well plate at 100 μL / well as the test compound well or control drug well; at 100 μL / well, the culture without the test compound or control drug As a negative control well. Place in a 37 ° C, 5% CO ₂ incubator and incubate for 72 hours.

Dissolve Resazurin (15mg / 50mL, 200 ×), methylene blue (25mg / 10mL, 1000 ×), potassium ferricyanide (0.329g / 100mL, 100 ×) and potassium ferrocyanide (0.422g / 100mL, 100 ×) Prepare 10 × Alamar Blue solution in PBS (0.1M, pH = 7.4), then dilute with phenol red-free DMEM medium to 1 × Alamar Blue solution, and prepare immediately before use.

The cells in the 96-well plate were carefully washed twice with PBS (0.1M, pH = 7.4), and the PBS was exhausted, and then added 1 × Alamar Blue solution at 100 μL / well; 100 μL 1 × Alamar Blue was added to the cell-free wells The solution serves as a blank control well. Incubate the 96-well plate at 37 ° C in a 5% CO ₂ incubator for 3 hours.

The fluorescence value of the cells was detected at 530nm at Ex 530 / Em with Victor X4 (Perkin Elmer). The fluorescence value of the test compound well is represented by F _{(test compound)} ; the fluorescence value of the blank control well is represented by F _{(blank control)} ; the fluorescence value of the negative control well is represented by F _{(negative control)} . Calculate the cell survival rate under different drug concentrations according to the following formula. Set 3 replicate wells at each concentration to obtain the average and standard deviation.

According to the cell survival rate, Prism Graph software was used to calculate the half-inhibitory concentration (IC ₅₀ ) of the test compound or the control drug on the cells.

3. Experimental results

The test results are shown in Table 4. The compounds significantly inhibited the growth of human colon cancer cell lines HCT116, HCT15, HT29 and COLO205. Among them, compounds 9, 19, 22 and 23 had better inhibitory effects.

Table 4. Half-inhibitory concentrations of test compounds on human colon cancer cell lines HCT116, HCT15, HT29, SW620 and COLO205 (IC ₅₀ , nM)

Example 11: Growth inhibitory activity of compounds 6, 22 and 23 on human colon cancer HT29 and HCT116 xenografts in nude mice

1. Test materials

1. Experimental animals

SPF grade BALB / c nude mice, female, are 6-8 weeks old at the start of administration and weigh 18-20 g. Provided by Changzhou Cavens Experimental Animal Co., Ltd. (Experimental Animal Production License: SCXK (Su) 2016-0010; Experimental Animal Use License: SYXK (Su) 2017-0007). Purchased animal certificate number: 201822212.

2. Test reagent

Polyethylene glycol 400 (PEG400), batch number 20180412, purchased from Chengdu Kelong Chemical Reagent Factory; sodium chloride injection (physiological saline), batch number A17111105, purchased from Hebei Tiancheng Pharmaceutical Co., Ltd .; DMSO, batch number Q6949, purchased from MP Biomedicals.

3. Cell lines

Human colon cancer HCT116 and HT29 cells were purchased from the Cell Resource Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences; HCT116 was cultured in 1640 medium containing 10% fetal bovine serum, and HT29 was cultured in 1640 medium containing 10% fetal bovine serum.

2. Test method

1. Preparation of the test compound

Low-dose group (administration dose: 4.0 mg / kg): Weigh 3.2 mg of the test compound before administration and dissolve it to 8 ml with a solvent (PEG400 50%, DMSO 5%, physiological saline 45%), the solution concentration is 0.4 mg / ml, administered intravenously, with a volume of 0.2ml / 20g body weight.

High-dose group (administration dose: 8.0 mg / kg): Weigh 6.4 mg of the test compound before administration, dissolve it to 8 ml with a solvent (PEG400 50%, DMSO 5%, physiological saline 45%), the solution concentration is 0.8 mg / ml, administered intravenously, with a volume of 0.2ml / 20g body weight.

2. Trial grouping and administration method

HCT116 and HT29 cells in the logarithmic growth phase were taken and inoculated under the aseptic conditions under the skin of the right armpits of 60 nude mice, respectively. The cell inoculation volume was 5 × 10 ⁶ cells / cell. Use a vernier caliper to measure the diameter of the transplanted tumor. When the tumor grows to about 100mm ³ , 56 HCT116 and HT29 tumor-bearing nude mice with good growth status and good tumor size uniformity are selected and randomly divided into 7 groups, 8 in each group. The model group, compound 6, 20 and 21 low-dose group and high-dose group. Each group was administered via tail vein injection, which was administered once a day on days 0, 1, 2, 3, and 4 for a total of 5 days. The model group was given an equal volume of vehicle control. Using the method of measuring tumor diameter, the anti-tumor effect of the test compound was dynamically observed. The tumor diameter was measured every two days and weighed at the same time. On the 20th day, the nude mice were sacrificed and the tumor masses were removed and weighed accurately.

The formula for calculating the tumor volume (TV) is:

Where a and b denote length and width, respectively.

The formula for calculating the% of tumor suppression is:

3. Test results

The test results are shown in Table 5, Table 6, and Figures 7 to 12. Compounds 6, 22, and 23, after being injected at a dose of 4.0 mg / kg and 8.0 mg / kg in tail vein for 5 consecutive days, can significantly Inhibit the growth of colon cancer cells HT29 and HCT116 in nude mice xenografts.

Table 5. Inhibitory effect of compounds on human colon cancer cell line HT29 xenograft tumor growth in nude mice (Mean ± SD, n = 8)

Table 6. Compound inhibitory effect on human colon cancer cell line HCT116 xenograft tumor growth in nude mice (Mean ± SD, n = 8)

Claims (10)

1. The use of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating or preventing gastric cancer or intestinal cancer,

   

   among them,

   R ¹ and R ² are independently selected from H, D, halogen, -CN, C _1-3 alkyl, substituted C _1-3 alkyl, C _1-3 alkoxy or substituted C _1-3 alkyl Oxy;

   R ^{3 is} selected from H, halogen, -OH, -CN, -C (= O) NH ₂ , substituted -C (= O) NH ₂ , C _1-3 alkyl, substituted C _1-3 alkyl, C _2-3 alkenyl, C _2-3 alkynyl, C _1-3 alkoxy or substituted C _1-3 alkoxy;

   R ^{4 is} selected from H, D, halogen, -OH, -CN, -NH ₂ , substituted -NH ₂ , -C (= O) NH ₂ , substituted -C (= O) NH ₂ , C _1-3 One or both of alkyl, substituted C _1-3 alkyl, C _2-3 alkenyl, C _2-3 alkynyl, C _1-3 alkoxy, or substituted C _1-3 alkoxy ;

   n = 1 or 2;

   R ^{5 is} selected from H, -CN, -OH, C _1-3 alkyl, C _1-3 alkoxy or amino acid residues;

   Z is selected from CH or N;

   The substituents in R ¹ , R ² , R ³ or R ⁴ are selected from D, halogen, OH, C _1-3 alkyl or C _1-3 alkoxy.
2. The use according to claim 1, wherein R ¹ and R ² are independently selected from H, D, F, Cl, -CN, -CH ₃ , -CF ₃ , -OCF ₃ or -OCHF ₂ .
3. The use according to claim 2, wherein R ^{1 is} selected from D, F, Cl, —CN, —CH ₃ or —CF ₃ , and R ^{2 is} selected from H.
4. The use according to claim 1, wherein R ^{3 is} selected from H, F, Cl, Br, -CN, -C (= O) NH ₂ , -CH ₃ , -CH ₂ CH ₃ , -CF ₃ ,- CH = CH ₂ , -C≡CH, -OCHF ₂ or -OCF ₃ .
5. The use according to claim 1, wherein R ^{4 is} selected from H, D, halogen, -CN, C _1-3 alkyl, substituted C _1-3 alkyl, C _1-3 alkoxy or substituted One or two of C _1-3 alkoxy groups.
6. The use according to claim 1, wherein R ^{4 is} selected from H, D, F, Cl, Br, I, -CN, -CH ₃ , -CF ₃ , -OCHF ₂ or -OCF ₃ ; R ^{5 is} selected from H or amino acid residues.
7. The use according to claim 1, wherein R ^{5 is} selected from H or 2,6-diamino-hexanoyl.
8. The use according to claim 1, wherein the compound is selected from:

   2-amino-5- (5-fluorobenzoselenazol-2-yl) benzonitrile,

   2-amino-3-fluoro-5- (5-fluorobenzoselenazol-2-yl) benzonitrile,

   2-ethynyl-4- (5-fluorobenzoselenazol-2-yl) aniline,

   5-fluoro-4- (5-fluorobenzoselenazol-2-yl) -2-methylaniline,

   2-bromo-6- (5-fluorobenzoselenazol-2-yl) pyridine-3-amine,

   3-amino-6- (5-fluorobenzoselenazol-2-yl) -2-carboxamidopyridine,

   4- (5-fluorobenzoselenazol-2-yl) -2-methylaniline,

   2-bromo-4- (5-fluorobenzoselenazol-2-yl) -6-methylaniline,

   2-bromo-6-fluoro-4- (5-fluorobenzoselenazol-2-yl) aniline,

   2,6-difluoro-4- (5-fluorobenzoselenazol-2-yl) aniline,

   2-fluoro-4- (5-fluorobenzoselenazol-2-yl) -6-methylaniline.
9. The use according to claim 1, wherein the compound according to claim 1, a pharmaceutically acceptable salt or a solvate thereof is used as an active ingredient or a main active ingredient, supplemented by a pharmaceutically acceptable auxiliary material to form a pharmaceutical combination Thing.
10. A method for treating human gastric cancer or intestinal cancer, characterized in that the compound of claim 1, a pharmaceutically acceptable salt, or a solvate thereof is administered to a person suffering from gastric cancer or intestinal cancer at 0.1-1000 mg per dose Or the pharmaceutical composition of claim 9 administered at a dose of 0.1-1000 mg per dose.

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